Myelopeptides and their therapeutic use

ABSTRACT

The present invention pertains to peptides comprising 4 to 10 amino acids including X 1 -Pro, Pro-X 2  or X 1 -Pro-X 2 , wherein X 1  and X 2  are independently selected from Lys, Arg, His, Asp, Glu, Asn and Gln, and the other amino acids are independently selected from Gly, Ala, Val, Leu, Ile, Nle, Nva, Pro, Phe, Tyr, Trp, Cys, Met, Ser and Thr. Such peptides, including LVCYPQ, FRPRIMTP, VVYPD and VDPP, are obtainable from porcine bone marrow cell culture, and have immunostimulating and anti-viral properties.

FIELD OF THE INVENTION

[0001] This invention relates to myelopeptides and their therapeuticuse.

BACKGROUND OF THE INVENTION

[0002] As reported by Mikhailova et al, Immun. Lett. 47:199-203 (1995),and in WO-A-9618652, the bone marrow cells of various animals and humansproduce a group of bioregulatory peptides named myelopeptides (MPs). MPshave a wide spectrum of functional activities: immunoregulatory,differentiating and opiate-like. They evoke 2-3-fold stimulation ofantibody production to various antigens and correct some immunedisorders. MPs influence the differentiation of bone marrow andperipheral blood cells derived from healthy and leukaemic donors. Theyinduce terminal differentiation in the leukaemic human HL-60 cell line,and show an effect on pain sensitivity.

[0003] More specifically, Mikhailova et al report two specifichexapeptides having immunoregulatory properties, i.e. FLGFPT (MP-1) andLVVYPW (MP-2). WO-A-9618652 discloses hexapeptides, having antitumouractivity, of the formula Y¹-Y²-Y³-Tyr-Pro-Trp. An example is MP-2.

SUMMARY OF THE INVENTION

[0004] Further peptides have been isolated from the supernatant ofporcine bone marrow cell culture by means of successive solid phaseextraction and HPLC. These novel peptides are of the type comprising 4to 10 amino-acids including Pro-X and/or X-Pro, X being a hydrophilicamino-acid, and which typically otherwise comprise hydrophobicamino-acids.

[0005] The novel peptides have therapeutic utility. For example, theymay be used where immunostimulating or antiviral activity is required.In particular, they may induce production of interferon(s), inhibitreplication of viruses, including HIV, thereby providing a protectiveeffect in mammals, and increase immune resistance towards bacterialinfection.

DESCRIPTION OF THE INVENTION

[0006] As indicated above, certain novel peptides have been obtainedfrom natural sources, using conventional procedures. They or otherpeptides of the invention may also be prepared by synthetic proceduresthat are known to those of ordinary skill in the art, e.g. the wellknown solid phase method.

[0007] Various preferred characteristics of the novel peptides aredefined in the claims. They may be tetra-, penta-, hexa-, hepta-, octa-,nona- or deca-peptides. Specific examples are LVCYPQ (herein MP-3),FRPRIMTP (MP-4), VVYPD (MP-5) and VDPP (MP-6), and data for thesepeptides are presented below. As will be evident from the results, thesepeptides do not have uniform characteristics. It will however also beevident that suitable tests can be made, to determine which is mostsuited for any particular application.

[0008] Peptides of the invention may be made up into pharmaceuticalformulations of any suitable type, using known carriers or diluents,e.g. as solutions or dispersions to be administered, with or without anadjuvant. The amount of the peptide that is administered will be chosenwith regard to factors such as the route of administration, the severityof the conditions, the age and health of the patient etc. The skilledphysician will be able to select appropriate amounts, e.g. based on theeffective dosages reported in the following results.

[0009] MP-3 Stimulates Macrophage Phagocytosis

[0010] Phagocytosis of opsonized sheep red blood cells (SRBC) by murineperitoneal macrophages was measured in the NBT-test (reduction of nitroblue tetrazolium by superoxide anions released by macrophages during anoxidative burst). Peritoneal cells were obtained from mice (CBAxC57B1)F1plated per well in 96-well flat-bottom plates (1×10⁶ cells/well) in 199medium. After 2 h incubation at 37° C. in an atmosphere of 5% CO₂ inair, non-adherent cells were removed by vigorous washing with warmedHank's balanced salt solution (BSS). 100 μl of NBT solution (1 mg/ml),50 μl 1% suspension of opsonized SRBC and MP-3, MP-1 or MP-2 atconcentrations from 10⁻⁶ to 10⁻¹⁸ g/ml were added to the wells. Controlwells had no MPs. After 1 hour incubation at 37° C., the cells werewashed with BSS and fixed with 10% formalin solution. 10 min later, thecells were washed with distilled water and dried. The insoluble blueformazan was solubilized by adding first 60 μl/well 2M KOH and then 70μl/well dimethyl sulphoxide (DMSO). The contents of the wells were thenmixed to complete solubilisation. The final solution had an intenseturquoise-blue colour. The OD₆₂₀ was read on an ELISA reader MultiskanMCC/340. The level of phagocytosis in each well treated with MPs wascompared to that in a control well (100%).

[0011] By contrast to MP-1 and MP-2, MP-3 stimulates macrophagephagocytosis in a dose-dependent manner. The dose curve has a bimodalcharacter. Maximal stimulation, up to 250%, occurs at doses of 10⁻⁸-10⁻⁷g/ml. There is one more peak of macrophage stimulation (at doses of10⁻¹⁶-19⁻¹⁷ g/ml). This effect is less pronounced but statisticallysignificant (p<0.05).

[0012] It may be concluded that the stimulation of macrophagephagocytosis by MP-3 results in its protective effect in infectedanimals.

[0013] MP-3 Increases the Survival of Mice Infected with Salmonellatyphimurium

[0014] MP-3 was used to inoculate (CBAxC57BL)F1 mice l/p at doses of0.5×10⁻⁴ g/mouse and 1×10⁻⁶ g/mouse. 24 hours later, these mice wereinfected with various doses of Salmonella typhimurium 415 (10², 10³, 10⁴or 10⁵ bacterial cells/mouse). The mice of a control group wereinoculated with saline solution. Each group contained 10 mice. The lifespan of each mouse was followed over 21 days.

[0015] A pronounced protective effect of MP-3 was obtained at both dosesused. At the level of 100% death in controls (10⁻⁵ and 10⁻⁴ bacterialcells/mouse), survival in the groups treated with MP-3 was 70-90%. Atthe level of 50% death in a control (10² bacterial cells/mouse), allmice treated with MP-3 stayed alive. This suggests that MP-3 protectsthe animals from bacterial infection due to its ability to stimulatemacrophage phagocytosis.

[0016] MPs Induce Resistance to Lethal Bacterial Infection

[0017] Acute bacterial infection was induced in laboratory mice of(CBAxC57B1)F1 origin using an intraperitoneal injection of 100 LD₅₀Salmonella thyphi which actually was 1,000 microbe bodies. The infectioncaused a rapidly progressing sepsis and death of 100% animals within 3days of such challenge.

[0018] Placebo control animals were injected intraperitoneally orsubcutaneously using 0.2 ml of 0.85% NaCl saline 24 h before challenge.Then these animals were subjected to the same procedure of infectionusing 100 LD₅₀ of S. thyphi. All the control animals died within 3 days,as if they were just challenged without any pre-treatment. By contrast,the pre-treatment of mice using intraperitoneal or subcutaneousinjection of MP-3, MP-4, MP-5 or MP-6, 24 h before the lethal challengeby 100 LD₅₀ S. thyphi, saved most of the animals. The MP's protectionwas dose-dependent.

[0019] The MPs, used at 1-10 μg doses per mouse, defended 90-100% ofanimals from subsequent challenge by the lethal dose of Salmonella. Bothroutes of injection, intraperitoneal and subcutaneous, were shown to bequite effective for pre-treatment using the MPs.

[0020] MP-4 Induces Terminal Differentiation in Leukaemia Cells

[0021] HL-60 cell line was obtained from bone marrow cells of patientswith acute myeloid leukaemia. These cells are myelomonoblasts which areintensively proliferating. They can differentiate to granuloid ormonocyte pathway only in the presence of appropriate stimulants.

[0022] The human myeloid HL-60 line was maintained in standard medium:RPMI-1640 medium supplemented with 15% (v/v) heat-inactivated fetal calfserum, 20 mM HEPES, 2 mM L-glutamine and 50 μg/ml gentamycin. Theinitial cell concentration was 2×10⁵ cells/ml. The cells were cultivatedat 37° C. in an atmosphere of 5% CO₂ in air. MP-4 was added to theculture at concentrations from 1×10⁻² to 1×10² g/ml. After 3 days ofcultivation, the cells were washed and reincubated in fresh culturalmedium. 3 days later, each culture was labelled with ³H-thymidine and¹⁴C-glycine, 4 hours before termination of culture. The cells wereharvested on the 6th day of cultivation and their DNA (³H) and protein(¹⁴C) radioactivity was measured. The mean counts per min (cpm) intriplicate culture were analyzed.

[0023] It is known that a decrease in chromosomal DNA synthesis and anincrease of total protein synthesis without histones are characteristicof the differentiation process. From changes of the ratio of ³H/¹⁴Cincorporation, it could be seen that MP-4 induces the differentiationprocess in HL-60 cells. The MP-4 effect on blastoid cells HL-60 isdose-dependent. The optimal dose is 0.1-5.0 μg/ml.

[0024] Morphological analysis of HL-60 cells treated with MP-4 confirmedthese results. There were about 60% mature forms (monocytes-macrophages)among blastoid cells. The differentiating effect of MP-4 was comparedwith that of the known differentiating factors phorbol myristate acetateand maturation inducer (T-lymphocyte differentiating factor). It may beconcluded that MP-4 induces terminal differentiation in leukaemia HL-60cells in the monocyte pathway.

[0025] Serum Interferon Increase Induced by MPs in Mice

[0026] Non-inbred white males mice aged 1.5-2 months, body weight 18-20g, were injected in the peritoneal cavity, with a single dose of MP-3,MP-4, MP-5 or MP-6: 0.01, 0.1, 1 or 10 μg (per mouse). Each of theexperimental groups of animals was represented by 25 mice receiving thesame dose of a preparation. 4, 24, 48, 72 or 96 h after injection, 5mice of each experimental group were sacrificed, their serum samplespooled and stored frozen at −60° C. until the interferon activity of thepooled serum measured. The latter measurement was performed by testingserum anti-viral activity in the in vitro cell cultures infected byencephalomyocarditis virus (EMV).

[0027] In more detail, cell cultures of L929 fibroblastoid cell linewere grown in alpha-MEM culture medium supplemented with 10% FBS.Cultures of 200,000 cells suspended in 0.1 ml of the culture medium wereplaced into wells of 96-well microplate and incubated in an atmosphereof 5% CO₂ at 37° C. The L929 cell cultures were infected using 100TCIC₅₀ dose of the established laboratory strain of EMV.Cytopathological damage in cell morphology was recorded 24 h frominfection.

[0028] Factor 2 serial dilutions of each serum sample were made usingalpha-MEM culture medium containing FBS. 0.1 ml of the designateddilution of serum sample was added into each microwell containing L929cell culture at the point of its infection with EMV.

[0029] On observing L929 cell damage caused by EMV within 24 h, thehighest dilution of a serum conferring 50% inhibition of the viralinfection was recorded. The reverse value of such highest dilution wastaken as an interferon titer of the serum represented in Units/ml.

[0030] In the control groups of mice, the strongest of known interferoninducers were used. Namely, Newcastle disease virus (NDS) and Ridostinwere used as a positive control to be compared with MPs by their seruminterferon-increasing activity.

[0031] Two phases of visible increase of early (4 h) and late (48 h)interferon were observed in the mouse serum after a single injection ofSerum interferon levels of 40 and 80 units/ml were reached, at 0.1 μgMP-4.

[0032] A single injection of 0.01 μg MP-5 led to a very strong increaseof late (48 h) serum interferon. The serum interferon titers reached 320units/ml, the level characteristic Ridostin, one of the strongestinterferon inducers yet known.

[0033] MP-3 and MP-6 weakly induced late interferon in the mouse serum.Levels of 20 and 40 units/ml, respectively, were achieved 48 h after asingle injection of 1-10 μg MP-3 and 0.1-1 μg MP-6.

[0034] Anti-viral Activity of the MPs in vitro

[0035] Being capable of inducing serum interferon increase in vivo, theMPs most probably induce synthesis of interferon in the appropriate cellculture in vitro. If so, this can be monitored according to thesecompounds' anti-viral action in cell culture in vitro, when infected byactively replicating virus.

[0036] In vitro culture of L929 mouse cells acutely infected byencephalomyocarditis virus (EMV) was used as a model to define activeconcentrations of MP-3, MP-4, MP-5 and MP-6 as related to theircapability to induce interferon synthesis in mammalian cells and henceblock viral infection in these cells. The L929 mouse fibroblastoid cellline was maintained in vitro as described above. MP-3, MP-4, MP-5 andMP-6 were added into triplicate cultures using the following finalconcentrations: 500, 250, 125, 63, 31, 16, 8, 4, 2, 1, 0.5, 0.25 μg per1 ml of culture. Ridostin was used as a control interferon-inducer.

[0037] 24 or 48 h post-injection, the L929 cell cultures were infectedby 100 TCID₅₀ of EMV. During the next 24 h the viral infection causeddramatic damages in L929 cells. When the cultures were pre-incubated inthe presence of MP-3, it did not prevent the cell layers from extensivedamage caused by subsequent virus infection using 100 TCID₅₀ of EMV.MP-5, by contrast, when added in a final concentration of 16 μg/ml ormore, completely abrogated destructive influence of the virus on L929cells. So did MP-6, when added at a concentration of 4 μg/ml or more.The anti-viral effect of MP-5 and MP-6 was quite comparable to that ofRidostin added at 0.5 μg/ml or more. MP-4 also defended the L929 cellsfrom the virus-induced damage, after 48 h exposure of the cells in thepresence of 32 μg/ml (or higher concentration) of the preparation beforetheir challenge by the virus.

[0038] Interferon Induction by MPs in Human Cells in vitro

[0039] The interferon-inducing capacities of the MPs was examined inboth L-41 human cell line and healthy donor peripheral blood cell (PBC)cultures. L-41 cells were maintained in medium 199 supplemented by 10%FBS and antibiotics. 200,000 cells per 1 ml of culture medium wereplaced in the wells of 24-well plastic culture plates and grown at 37°C. in the atmosphere of 5% CO₂/ When a cell monolayer was formed, thecontrol and experimental interferon-inducers were introduced in thecultures in the centration desired. 24 h after interferon induction, theculture supernatants were harvested and then examined for interferoncontent.

[0040] The healthy donor PBC were incubated in the wells of 96-wellculture plates and the culture supernatants were harvested 24 h afterthe test preparation was added to the culture.

[0041] To measure the interferon activity of the supernatants harvested,serial dilutions of the supernatants were prepared and then added intofresh L-41 cultures infected using 100 TCID₅₀ of EMV. The interferontiter of the supernatant was recorded, as described above for interferontitration of the mouse serum in cultures of mouse L929 cells.

[0042] 1 μg/ml MP-3 induced an intensive production of interferon byhuman cells, both L41 and donor PBC. 0.01 μg/ml MP-4 showed induction ofinterferon synthesis comparable to MP-3 in L-41 cells. MP-5 at smalldoses (0.001 μg/ml induced interferon in donor PBC, and at larger doses(1 μg/ml) was active with L-41 cells. MP-6 induced relatively smallamounts of interferon in both L-41 and donor PBC.

[0043] HIV-Inhibition by MPs in vitro

[0044] The anti-viral activity of MP-3, MP-4, MP-5 and MP-6 was examinedusing in vitro culture of MT4 cells, a human T-lymphoblastoid cell lineinfected by HIV. As a source of infective virus, the laboratory HT HIV27 virus strain was used. It was capable of causing chronicHIV-infection, with the production of non-damaged HIV-1 infectiveparticles in MT4 cells in vitro.

[0045] The intensity of HIV infection and the respective anti-HIVinfluence of the MPs was evaluated according to the following criteria:

[0046] (a) alterations of normal MT4 cell morphology induced by HIV(cytopathogenic effect);

[0047] (b) HIV antigens found in infected MT4 cells using luminescencemicroscopy of the cells pre-labelled with fluorescent anti-HIVantibodies;

[0048] (c) HIV proteins detected in HIV-infected MT4 cell culture mediumusing immunofluorescence.

[0049] The HIV-infected MT4 cells were incubated in RPMI-1640 culturemedium supplemented with 10% FBS and 2 mM L-glutamine. The MT4 cells(0.5 million cells per 1 ml of culture medium) were placed in wells of a24-well plastic culture plates and kept for 7 days of 37° C. in anatmosphere of 4.5% CO₂. Each of the MPs examined was added intorespective wells of the culture plates, to final concentrations of 0.1,0.5, 1, 5, 10, 50 and 100 μg per 1 ml of culture were used.Azidothymidine (Sigma Chemical Co.) at its final concentration of 0.1μg/ml, was used as a positive anti-viral control during each of theexperiments.

[0050] Standard cell viability tests performed 24 h from cultivationshowed MP-3, MP-4, MP-5 and MP-6 caused no visible damage of MT4 cellsgrowing in vitro. Of these four compounds, the most pronounced anti-HIVactivity was observed with MP-3. In the presence of 1-5 μg/ml or higherconcentration of MP-3, HIV replication was completely stopped: nocytopathogenic effect or production of viral protein was recorded insuch cultures. 50% inhibition of HIV replication was observed in thepresence of 10 μg/ml MP-4. MP-5 and MP-6 did not influence on HIVreplication in MT4 cell cultures in vitro. Azidothymidine (0.1 μg/ml)caused 100% inhibition of HIV replication in the infected MT-4 cells.

1 6 1 6 PRT Sus species 1 Phe Leu Gly Phe Pro Thr 1 5 2 6 PRT Susspecies 2 Leu Val Val Tyr Pro Trp 1 5 3 6 PRT Sus species 3 Leu Val CysTyr Pro Gln 1 5 4 8 PRT Sus species 4 Phe Arg Pro Arg Ile Met Thr Pro 15 5 5 PRT Sus species 5 Val Val Tyr Pro Asp 1 5 6 4 PRT Sus species 6Val Asp Pro Pro 1

We claim:
 1. A peptide comprising 4 to 10 amino acids including X¹-Pro,Pro-X² or X¹-Pro-X², wherein X¹ and X² are independently selected fromLys, Arg, His, Asp, Glu, Asn and Gln, and the other amino acids areindependently selected from Gly, Ala, Val, Leu, Ile, Nle, Nva, Pro, Phe,Tyr, Trp, Cys, Met, Ser and Thr.
 2. The peptide according to claim 1,which comprises predominantly other amino acids as defined in claim 1.3. The peptide according to claim 1, wherein the N-terminal comprises 1,2, 3 or 4 amino acids independently selected from Gly, Ala, Val, Leu,Ile, Nle, Nva, Phe, Tyr, Trp, Cys and Met.
 4. The peptide according toclaim 1, including Pro-X².
 5. The peptide according to claim 1, whereinX² is a terminal amino acid.
 6. The peptide according to claim 1,including X¹-Pro.
 7. The peptide according to claim 6, includingZ-X¹-Pro, wherein Z is terminal and one of the other amino acids.
 8. Thepeptide according to claim 1, which is or comprises LVCYPQ (SEQ ID NO.3).
 9. The peptide according to claim 1, which is or comprises FRPRIMTP(SEQ ID NO. 4).
 10. The peptide according to claim 1, which is orcomprises VVYPD (SEQ ID NO. 5).
 11. The peptide according to claim 1,which is or comprises VDPP (SEQ ID NO. 6).
 12. The peptide according toclaim 1, free from other components of bone marrow.
 13. The peptideaccording to claim 1, in a pharmaceutically acceptable carrier ordiluent.
 14. The peptide according to claim 2, wherein the N-terminalcomprises 1, 2, 3 or 4 amino acids independently selected from Gly, Ala,Val, Leu, Ile, Nle, Nva, Phe, Tyr, Trp, Cys and Met.
 15. A method forthe treatment or prophylaxis of a viral or bacterial infection, or adisease requiring immunostimulation, in a patient, said methodcomprising administering an effective amount of a peptide of claim 1 tothe patient.